Publications and other references referred to herein are incorporated herein by reference and are numerically referenced in the following text and respectively grouped in the appended Bibliography which immediately precedes the claims.
The present invention relates to the chemical modification of aminoglycans, proteins and amine coated surfaces by means of the covalent bonding of polymer chains of polyoxyalkylenes, such as polyethylene oxide (also called polyethyleneglycol) and polypropylene glycol.
Polyethylene glycol (PEG) use in biotechnology and biomedical applications continues to expand and has recently been reviewed (1). Modification of enzymes (2), RGD peptides (3), liposomes (4), and CD4-IgG glycoprotein (5) are some of the recent advances in the use of polyethylene glycol. The modification of toxicity, pharmacokinetics, biodistribution and other biofunctions are a number of the promising areas for the use of this simple polymer. Surfaces treated with PEG have been shown to resist protein deposition and have improved resistance to thrombogenicity when coated on blood contacting biomaterials (6). Accordingly, application of PEG based coatings to various polymeric materials especially with respect to "continuous" coating of microporous hollow fiber or other plastic parts would be very useful for medical devices.
Electrophilic activated polyoxyalkylenes such as PEG for continuous coating applications should satisfy the following requirements:
1. The rate of reaction between an amino group coated surface and/or an amino containing biomolecule with an electrophilically activated PEG should have an fast reaction rate under mild conditions. PA1 2. Ideally, the electrophilically activated PEG should have an appropriate hydrolysis half-life in water at pH values of 7.5-8.5. This is especially important with respect to polymeric substrates to be coated that can not withstand exposure to organic solvents. PA1 3. Formation of a covalent bond between an amino-containing biomolecule and the electrophilically activated PEG should be demonstrated by spectroscopic means such as nuclear magnetic resonance an/or infrared spectroscopy to demonstrate that the chemistry proceeds as expected. PA1 4. Should organic solvents be used in the coating process, stability of the electrophilically activated PEG should be appreciable for economical reasons. PA1 5. Reaction of the electrophilically activated PEG with the biological molecules of interest should be site directed so that crucial receptor and/or active sites are not blocked. In turn, retained biological function should be demonstrated by an appropriate assay. PA1 6. Quality or functionality of the electrophilically activated PEG should be easily determined by rapid spectroscopic means during a manufacturing process. PA1 7. The leaving group released upon acylation of amino groups should have high solubility in the reaction medium, minimal adsorption to the modified substrate and be non-toxic ideally. PA1 8. The electrophilically activated PEG should have long term stability for shelf life storage purposes. PA1 9. The covalent bond formed should be hydrolysis resistant under both the coating conditions and subsequent "actual use" conditions.
In order to covalently bond, the hydroxyl group of PEG must be "activated". This has been reported as accomplished by the use of a number of reactive functional groups including cyanurylate (7-9), tresylate (10-11), N-hydroxysuccinimide derived active esters (12-17), carbonates (18-20), imidazolyl formates (21-22), 4-dithiopyridines (23), isocyanates (24) and epoxides (25). Each of the above functional groups possess disadvantages which range from leaving group toxicity, conjugates that are prone to hydrolysis under physiological conditions and slow reaction rate in the conjugation process. Radiolabeled urethane-PEG derivative stability has been demonstrated under a variety of physiological conditions (26). The hydrolysis resistant urethane bond produced by sufficiently reactive PEG carbonates may offer considerable advantage in avoiding hydrolysis of the conjugation covalent bond. To date, literature reports on the use of PEG carbonates have focused on the modification of protein or polypeptides. Driven by our interest in developing a continuous coating process for covalently bound heparin on a microporous hollow fiber surface, we explored the reactivity of various PEG carbonates with the aminoglycan D-glucosamine and several commercially available sodium heparins.